This invention relates to methods for detecting Pneumocystis carinii infection in humans, specifically to such methods that involve polymerase chain reaction or other amplification of nucleic acid sequences that encode a Pneumocystis carinii sp. f. hominis protein.
Pneumocystis carinii is an important life threatening opportunistic pathogen of immunocompromised patients, especially those with human immunodeficiency virus (HIV) infection. Conventional diagnosis of Pneumocystis carinii pneumonia (PCP) involves analysis of a tissue sample or oropharyngeal secretion sample for the presence of a P. carinii organism through staining and microscopic examination. Sample acquisition techniques have included such invasive methods as transbronchial biopsy, percutanenous lung biopsy, or open lung biopsy. Each of these techniques is fraught with possible complications and requires significant time and expense. In the mid 1980""s, bronchoalveolar lavage (BAL) was introduced as a less invasive, less expensive, and less complication-prone technique for acquiring samples to be used in PCP diagnosis (Ognibene et al. (1984) Am. Rev. Respir. Dis. 129:929-932). However BAL, coupled with bronchoscopy, still required special equipment and facilities, as well as the time of a physician and technician. Simpler still, it is now known that the Pneumocystis organism can also be detected in induced sputum samples (Bigby et al. (1986) Am. Rev. Respir. Dis. 133:515-518; Kovacs et al. (1988) NEJM 318:589-593).
Advances also have occurred in the techniques used to detect the Pneumocystis organism in tissue and oropharyngeal secretion samples. Direct microscopic examination of clinical samples stained with, for instance, Giemsa stain or toluidine blue O, requires time-consuming sample preparation and subsequent examination by specially trained and experienced microscopy technicians (see, for instance, Bigby et al. (1986) Am. Rev. Respir. Dis. 133:515-518). This procedure has been somewhat simplified and rendered more amenable to mechanization through the use of monoclonal antibodies in detection of P. carinii antigens in clinical samples (Kovacs et al. (1988) NEJM 318:589-593). A few groups have used oligonucleotide probes complementary to P. carinii nucleotide sequences to detect the organism through hybridization, as in U.S. Pat. No. 5,164,490 (the Santi patent).
Polymerase chain reaction (PCR) -mediated amplification of DNA or RNA-encoding sequences has been used to diagnose various diseases including leprosy (Santos et al. (1997) J. Med. Microbiol. 46:170-172) and PCP. This technique exhibits increased sensitivity over simple probe hybridization methods. Primers complementary to sequences encoding P. carinii mitochondrial or chromosomal ribosomal RNA (rRNA) have been used to amplify Pneumocystis-specific DNA sequence, as in Wakefield et al. (1990) Mol. Biochem. Parasit. 43:69-76; Wakefield et al. (1990) Lancet 336:451453; Lipschik et al. (1992) Lancet 340:203-206; WO 91/19005; and U.S. Pat. Nos. 5,519,127 (the Shah patent), 5,593,836 (the Niemiec patent) and 5,776,680 (the Leibowitz patent).
Other recent research advances relate to elucidating the molecular mechanisms involved in P. carinii infection. A great deal of interest has focused on the major surface glycoprotein (MSG; also called glycoprotein A) of P. carinii, because it is considered to be both a virulence factor and a target of host immune responses. MSG is the most abundant protein expressed on the surface of P. carinii, as assessed by Coomassie blue staining. It appears to play a critical role in the pathogenesis of pneumocystosis, possibly by acting as an attachment ligand to lung cells. MSG is also a target of both humoral and cellular immune responses by the host.
Multiple genes encode the MSG of rat-P. carinii, and different MSGs may be expressed in the lung of a rat infected with P. carinii (Angus et al. (1996) J. Exp. Med. 183:1229-1234; Kovacs et al. (1993) J Biol. Chem. 268:6034-6040). Similarly, multiple genes encode the MSG of P. carinii infecting ferrets and mice (Haidaris et al. (1998) DNA Res. 5:77-85; Haidaris et al. (1992) J. Infect. Dis. 166:1113-1123). Additional studies have shown that there is a single genomic site for expression of rat MSG variants (Edman et al. (1996) DNA Cell Biol. 15:989-999; Sunkin and Stringer (1996) Mol. Microbiol. 19:283-295; Wada and Nakamura (1996) DNA Res. 3:55-64; Wada et al. (1995) J. Infect. Dis. 171:1563-1568). These studies suggest that P. carinii has developed an elaborate system for antigenic variation, presumably to evade host defense mechanisms.
Molecular and immunological studies have clearly demonstrated that P. carinii isolated from different host species are distinct organisms, and may in fact be separate species (Gigliotti (1992) J. Infect. Dis. 165:329-336; Keely et al. (1994) J. Eukaryot. Microbiol. 41:94S; Kovacs et al. (1989) J. Infect. Dis. 159:60-70; Stringer (1993) Infect. Agents Dis. 2:109-117). There is a high level of variation among orthologous genes, including the MSG genes, isolated from different host-specific strains of the Pneumocystis. Hence, diagnosis of P. carinii infection in human patients ideally requires P. carinii sp. f. hominis (hereinafter xe2x80x9chuman-P. cariniixe2x80x9d) derived reagents.
The cloning of human-P. carinii MSG genes has recently been reported (Garbe and Stringer (1994) Infect. Immun. 62:3092-3101; Stringer et al. (1993) J. Eukaryot. Microbiol. 40:821-826); however, only one full-length sequence was reported.
The inventors have discovered that human-P. carinii MSG is encoded for by a large, highly-conserved gene family, with a particularly conserved region of about 100 amino acids in the C-terminal region of the proteins. The have further discovered that direct detection or nucleic acid amplification (e.g., PCR amplification) of human-P. carinii MSG-encoding genes provides a particularly sensitive and specific technique for the detection of P. carinii, and the diagnosis of PCP.
This invention encompasses the purified novel human-P. carinii proteins represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14, and isolated nucleic acid molecules that encode these proteins. Specific nucleic acid molecules encompassed in this invention include those represented in SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4, SEQ ID NO: 5; SEQ ID NO: 6, SEQ ID NO: 7; SEQ ID NO: 15; and SEQ ID NO: 17. Also encompassed within this invention are the isolated nucleic acid sequences that encode the carboxy-terminal conserved about 100 amino acids of the disclosed human-P. carinii MSGs; these may be used for amplification or as probes. The sequences of these conserved nucleic acid molecule regions include residues 2794-3042 of HMSGp1 (SEQ ID NO: 1), 2758-3006 of HMSGp3 (SEQ ID NO: 3), 2845-3090 of HMSG11 (SEQ ID NO: 5), 2839-3084 of HMSG14 (SEQ ID NO: 7), 2836-3081 of HMSG32 (SEQ ID NO: 9), 2809-3054 of HMSG33 (SEQ ID NO: 11), 2821-3072 of HMSG35 (SEQ ID NO: 13), or 1-249 of HMSGp2 (SEQ ID NO: 15). In addition, this invention encompasses sequences with at least 70% sequence identity to these regions, and recombinant vectors comprising such nucleic acid molecules and conserved regions from within such nucleic acid molecules, as well as transgenic cells including such a recombinant vector.
Another aspect of this invention provides a method of detecting the presence of Pneumocystis carinii in a biological specimen, by amplifying with a nucleic acid amplification method (e.g., the polymerase chain reaction) a human-P. carinii nucleic acid sequence using two or more oligonucleotide primers derived from a human-P. carinii MSG protein encoding sequence, then determining whether an amplified sequence is present. In a preferred embodiment of this invention, the human-P. carinii nucleic acid sequence is a highly conserved region within an MSG-protein encoding sequence. Such a highly conserved region may, for instance, include residues 2794-3042 of HMSGp1 (SEQ ID NO: 1), 2758-3006 of HMSGp3 (SEQ ID NO: 3), 2845-3090 of HMSG11 (SEQ ID NO: 5), 2839-3084 of HMSG14 (SEQ ID NO: 7), 2836-3081 of HMSG32 (SEQ ID NO: 9), 2809-3054 of HMSG33 (SEQ ID NO: 11), 2821-3072 of HMSG35 (SEQ ID NO: 13), or 1-249 of HMSGp2 (SEQ ID NO: 15). A further aspect of this invention is the method of detecting the presence of Pneumocystis carinii in a biological specimen, by determining whether an amplified sequence is present, for instance by electrophoresis and staining of the amplified sequence, or hybridization to a labeled probe of the amplified sequence. Appropriate labels for the hybridization probe include a fluorescent molecule, a chemiluminescent molecule, an enzyme, a co-factor, an enzyme substrate, or a hapten. The nucleotide sequence of such a probe can be chosen from any MSG gene sequence that is amplified in the detection method, and for instance can include a nucleic acid sequence according to SEQ ID NO: 19.
Another aspect of this invention is a method of detecting the presence of Pneumocystis carinii in a biological specimen by exposing the biological specimen to a probe that hybridizes to a human-P. carinii nucleic acid sequence derived from a human-P. carinii MSG protein encoding sequence. The labeled probe to be used in this method may, for instance, include the nucleic acid sequence of SEQ ID NO: 19.
This invention also encompasses one or more oligonucleotide primers including at least 15, or at least 20, 25, 30, 35, 40, 50, or 100, contiguous nucleotides from any of the highly conserved regions within an MSG-protein encoding sequence disclosed herein, or from any nucleic acid sequences having at least 70%, or at least 90% or 95%. sequence homology with these sequences. Specific examples of such oligonucleotide primer sequences are shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23, and SEQ ID NO: 24. Of these primers, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO:23 may serve as upstream primers, while SEQ ID NO: 20 and SEQ ID NO: 24 may serve as down stream primers.
Kits for detection of a human-P. carinii nucleic acid sequence are another aspect of this invention. Such kits may include at least a pair of primers each comprising at least 15, or at least 20, 25, 30, 35, 40, 45, 50, or 100 contiguous nucleotides of any of the conserved regions of the herein disclosed MSG-encoding sequences, and homologs having at least 70% identity with such sequences. Representative primers include those represented by the nucleotide sequences of SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; and SEQ ID NO: 24. These kits may further including a positive nucleic acid amplification (e.g., PCR) control sequence.
Antibodies raised to the peptide sequence according to SEQ ID NO: 25 or SEQ ID NO: 26 are also included within the scope of this invention.
The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description of several embodiments. which proceeds with reference to the accompanying figure and tables.